Controversy still persists as to success of multi-tissue differentiation from a transplanted progenitor (stem) cell population either due to differences in tissue source and cell preparation and to methods used to judge the level of differentiation. Increasingly it is appreciated that engraftment based on standard histological criteria or expression of a ubiquitous genetic marker may not be validated when a tissue specific donor expressed marker gene is utilized. We concur with this opinion based on our experience using osteoblast restricted GFP reporter transgenic mice in a variety of transplantation protocols. We can find no evidence for osteoblast engraftment by cells in a total bone marrow transplant or when marrow stromal cells are injected systemically. However there is robust osteoblast differentiation and incorporation into host bone when the stromal cells are administered in the intramedullary space. Using this type of transplantation protocol, four distinguishable colors of GFP transgenic mice and a rapid, sensitive and specific histological method for assessing osteoblast engraftment, we will map the position of an osteoprogenitor cell population to a hypothesized lineage map that originates from a hemangioblast. This adult progenitor cell has the properties of hematopoietic, endothelial and osteoblast differentiation and is hypothesized to have sufficient self-renewal potential to engraft and support new bone formation for a clinically significant period of time. Evidence for this type of cell in five clinically relevant tissue sources (bone marrow, bone tissue, muscle, fat and newborn umbilical cord) will be generated and tested for in vivo osteoblast differentiation optimal for new bone formation. Methods to identify and enrich for the cells responsible for optimal osteogenesis will be pursued by FACS sorting based on surface antigens or expression of promoter - GFP transgenes active during early lineage development. Microarray studies of the enriched population may suggest other strategies to further enrich for the optimal progenitor cells. This grant will provide a head to head comparison of the osteoprogenitor potential for bone transplantation of cells from 5 different tissues. It will assess the effectiveness of enrichment protocols and develop a strategy to relate the position of the progenitor cells to a hypothesized but testable map of lineage progression from a true embryonic stem cell to a cell with sufficient adult stem cell properties to be useful in cell and gene therapy applications for diseases of bone.